CN101338337B - Polymorphism site genotype estimation depression, use and process of medicine effect and kit - Google Patents
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Abstract
The present invention relates to a 5-HTT gene 13C/T polymorphism site genotype used for forecasting an individual depression sickness degree as well as the sickness degree of the core symptom of the depression, forecasting the effect of an antidepressant as well as the usage, the method and the reagent box of the effect of the antidepressant on the core symptom of the depression; wherein, when the 5-HTT gene 13C/T polymorphism site genotype is of 13TT homozygous type, the individual depression sickness degree and the sickness degree of the core symptom of the depression are forecasted to be more serious; the effect after taking the antidepressant is better; the effect of the antidepressant to the core symptom of the depression is better; when the 5-HTT gene 13C/T polymorphism site genotype is of 13CC homozygous type or 13CT heterozygote type, the individual depression sickness degree and the sickness degree of the core symptom of the depression are forecasted to be lighter; the effect after taking the antidepressant is poorer; the effect of the antidepressant to the core symptom of the depression is poorer.
Description
Technical field
The pleomorphism site genotype that the present invention relates to five hydroxytryptamine translocator (5-HTT) gene is used to predict the purposes of the ill degree of dysthymia disorders, thymoleptic drug effect.The invention still further relates to a kind of by measuring the pleomorphism site genotype of 5-HTT gene, the method and the test kit of the ill degree of prediction dysthymia disorders, thymoleptic drug effect.The invention belongs to field of medicaments.
Background technology
Dysthymia disorders (Major depression) is to serve as main performance and with the affective disorder of other psychology and somatization with spiritual psychological syndromes such as depressed, anhedonia and hebetudes.The clinical manifestation of dysthymia disorders is widely different, and its main clinical performance can be divided into core symptom and simultaneous phenomenon (mental symptoms group and somatization group) two big classes.Depressed core symptom comprises mental state or depressed, interest blank and enjoyment forfeiture, and this is depressed key symptoms.Wherein, the depressed patient of being meant to experience mood low, sad; Interest blank is meant that patient lacks enjoyment to the activity of liking before various; The enjoyment forfeiture is meant that patient can't experience enjoyment from life, or day anhedonia (anhedonia).More than three kinds of masters levy and connect each other, can occur simultaneously or only with wherein a certain, two kinds outstanding.
Most depressive patients are attended by tangible anxiety symptom and somnopathy etc., also can become the main suit that the patient gives prominence to sometimes.Anxiety symptom shows as two types of subjective anxiety and somatic anxieties.Subjective anxiety is shown as anxiety, anxiety, fears, frightened, to the baffled worry in future etc.; Symptoms such as somatic anxiety is shown as palpitaition, breathes hard, perspiration, dry, frequent micturition.That the patient of serious anxiety can show is intense, irritability, causeless ignition, in addition occur hurting sb.'s feelings, behavior such as self inflicted injury.The symptom of anxiety usually is associated with somnopathy, mainly shows as the disappearance of difficulty falling asleep and sleep sense.Depressive symptom and anxiety symptom usually merge existence, and become the common sympton that constitutes the depressive symptom group with somnopathy.
The pathogeny of dysthymia disorders it be unclear that, and thinks that at present monoamine (comprising catecholamine and five hydroxytryptamine etc.) class material plays a significant role therein.There are confidential relation in the generation and the development of serotonin (5-HT) level or changing function and dysthymia disorders.Be positioned at serotonin transporter (the 5-hydroxytryptamine transporter on the neuronic presynaptic membrane of 5-HT, (Meyer JH 5-HTT) plays an important role in the 5-HT system, Houle S, Sagrati S.et al.Brain serotonin transporter bindingpotential measured with carbon 11-labeled DASB positron emissiontomography:effects of major depressive episodes and severity ofdysfunctional attitudes.Arch Gen Psychiatry.2004; 61:1271-9).5-HTT combines with 5-HT in the synaptic cleft, and it is transported to neuronic axon ends.5-HTT regulates 5-HT removal and 5-HT release cycle fast after the nerve stimulation.5-HTT is positioned at the nerve ending presynaptic membrane, near the 5-HT neurone dendron and the projected area thereof of midbrain, brain stem nuclei of median raphe, and as hypothalamus, limbic system, Basal ganglia and pallium, particularly prefrontal lobe have 12 hydrophobic transmembranes (TM) zone.Human 5-HTT gene name is called SCL6A4, and (solute carrier family 6 is the neurotransmitter translocator, and the member 4, solute carrier family6, member4), be positioned at 17q11.1-17q12, cross over 37.8kbp, comprise 14 exons, 630 the amino acid whose albumen of encoding.5-HTT gene transcription activity is regulated by the regional called after serotonin of the 1.4kb5 ' that is positioned at transcripting start point upstream T promoter region (5-HTTLPR), is inserted and lacked by 44bp to form l and two kinds of allelotrope of s.Three variations are arranged in the untranslated zone: 1 is positioned at the 380bp disappearance between 5-HTTLPR and transcription initiation site, unstable in vivo, may relate to the 5-HTTLPR dependent mechanism.2VNTR (the variable number polyphone repeats): the polyphone that is positioned at the variable number in the 2nd intron zone repeats.The transversion of the adenylic acid (AMP) site G-T in 33 ' UTR zone.
Dysthymia disorders is the polygenic inheritance disease, has very high biology heterology.Almost completely rely on doctor's clinical experience for a long time for the diagnosis and treatment of dysthymia disorders, subjectivity is strong, the objective biology sign of shortage, makes the diagnoses and treatment level of dysthymia disorders lag behind other physical diseases.Simultaneously, conventional anti-depressant therapy only about 1/2-2/3 patient has clinical efficacy, what can reach clinical remission more is low to moderate 1/3-1/2, and the antidepressant drug clinical onset of action is slow, usually take medicine and just can give full play to antidepressant effect (Berman RM after 4-6 weeks, Narasimhan M, Charney DS:Treatment-refractory depression:definitionsand characteristics.Depress Anxiety.1997; 5:154-164), and different patient uses with very big difference is arranged in its curative effect of a kind of medicine and the side reaction.These problems are perplexing the clinician for a long time.
The single nucleotide polymorphism (SNP) of research carry out to(for) the complicacy disease provides important foundation for the pathogeny of understanding disease, the diagnosis and the disease-susceptible humans Journal of Sex Research of disease.All there is SNP in 93% Human genome, is generally diallele, is the best genetic marker of application prospect.The variation of genomic dna sequence is considered to determine people's the disease susceptibility and the important factor of drug reaction.In order earlier more fully to prevent and treat dysthymia disorders, press for for dysthymia disorders and pharmacological agent thereof and carry out early prediction, the present invention promptly provides a kind of new ill severity of prediction dysthymia disorders and purposes, method and the test kit of pharmacological agent.
Summary of the invention
Technical problem to be solved by this invention is by the research gene polymorphism sites genotype relevant with the ill severity of dysthymia disorders, provides the pleomorphism site genotype of 5-HTT gene to be used to predict the purposes of dysthymia disorders severity.
Simultaneously, the pleomorphism site genotype that the invention provides the 5-HTT gene is used to predict the purposes of dysthymia disorders core symptom severity.
In the present invention, " dysthymia disorders " is meant with depressed, and spiritual psychological syndromes such as anhedonia and hebetude are the affective disorder of main performance and and somatization psychological with other.The clinical manifestation of dysthymia disorders comprises core symptom and simultaneous phenomenon two big classes.Depressed core symptom comprises depressed (or mental state is low), interest blank and enjoyment forfeiture.Wherein, it is low that listless patient experiences mood, sadness; Interest blank is meant that patient lacks enjoyment to the activity of liking before various; The enjoyment forfeiture is meant that patient can't experience enjoyment from life, or says anhedonia (anhedonia).Anxiety and somnopathy are the simultaneous phenomenons of dysthymia disorders.
Another technical problem that will solve of the present invention provides the purposes of the polymorphism site genotype estimation antidepressant drug drug effect of 5-HTT gene.
Simultaneously, the present invention also provides the pleomorphism site genotype of 5-HTT gene to be used to predict the purposes of antidepressant drug to dysthymia disorders core symptom drug effect.
In the present invention, " antidepressant drug " is meant and is selected from serotonin reuptake inhibitor (selectiveserotonin reuptake inhibitors, SSRI), selectivity 5-HT and norepinephrine (NE) reuptake inhibitor (serotonin-norepinephrine reuptake inhibitor, SNRI), 5-HT receptor antagonist/reuptake inhibitor (serotonin antagonist/reuptake inhibitor, SARI), NE and specificity 5-HT energy thymoleptic (noradrenergic and specificserotonergic antidepressant, NaSSA).Wherein, SSRI class medicine comprises fluoxetine (fluoxetine), paroxetine (Paroxetine), Sertraline (Sertraline), fluvoxamine (Fluvoxamine), citalopram (Citalopram) and S-citalopram (Escitalopram).SNRI class medicine comprises Venlafaxine (venlafaxine) and duloxetine (duloxetine).SARI class medicine comprises trazodone (Trazodone), nefazodone (Nefazodone).NaSSA class medicine comprises mirtazapine (Mirtazapine).Wherein, the preferred SSRI class of antidepressant drug medicine is more preferably the fluoxetine in the SSRI class medicine.
In the present invention, the pleomorphism site genotype of 5-HTT gene preferred " the 13C/T pleomorphism site genotype of 5-HTT ", the 13C/T pleomorphism site genotype of 5-HTT " be meant that serotonin transporter gene (SCL6A417q11.1-17q12) is positioned at the pleomorphism site genotype at 13 intron places; there is the single nucleotide polymorphism (NCBI SNP ID:rs2054847) of C/T in this site; promptly when two karyomit(e) corresponding positions were " C ", this loci gene type was " 5-HTT13CC is homozygous "; When two karyomit(e) corresponding positions were respectively " C " and " T ", this loci gene type was " a 13CT heterozygous "; When two karyomit(e) corresponding positions were " T ", this loci gene type was " 13TT is homozygous ".
In the present invention, the genotype polymorphism of 5-HTT is also further by determining to exist the genotype of other pleomorphism site of linkage disequilibrium to realize with it near the 13C/T pleomorphism site.Preferably, described other pleomorphism site comprises nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position.
In one embodiment of the invention, when:
(1) 5-HTT13C/T (rs2054847) pleomorphism site genotype is 13TT when homozygous, predicts that individual dysthymia disorders severity is heavier;
When (2) 5-HTT13C/T (rs2054847) pleomorphism site genotype is the homozygous or 13CT heterozygous of 13CC, predict that individual dysthymia disorders severity is lighter.
In embodiment provided by the invention, when:
(1) 5-HTT13C/T (rs2054847) pleomorphism site genotype is 13TT when homozygous, predicts that individual dysthymia disorders core symptom severity is heavier;
When (2) 5-HTT13C/T (rs2054847) pleomorphism site genotype is the homozygous or 13CT heterozygous of 13CC, predict that individual dysthymia disorders core symptom severity is lighter.
In another embodiment provided by the invention, when:
(1) 5-HTT13C/T (rs2054847) pleomorphism site genotype is 13TT when homozygous, and the drug effect of prediction thymoleptic is better;
When (2) 5-HTT13C/T (rs2054847) pleomorphism site genotype was the homozygous or 13CT heterozygous of 13CC, the drug effect of prediction thymoleptic was relatively poor.
In embodiment provided by the invention, when:
(1) 5-HTT13C/T (rs2054847) pleomorphism site genotype is 13TT when homozygous, and the prediction thymoleptic are better at the drug effect of dysthymia disorders core symptom;
When (2) 5-HTT13C/T (rs2054847) pleomorphism site genotype was the homozygous or 13CT heterozygous of 13CC, the prediction thymoleptic were relatively poor at the drug effect of dysthymia disorders core symptom.
The present invention also provides by measuring from 5-HTT gene 13C/T pleomorphism site genotype in experimenter's the biological sample, predicts the ill severity of individual dysthymia disorders, the ill severity of dysthymia disorders core symptom, the drug effect of predicting thymoleptic and the thymoleptic method to the drug effect of dysthymia disorders core symptom.Wherein, when 5-HTT13C/T (rs2054847) pleomorphism site genotype is that 13TT is when homozygous, predict that the ill degree of individual dysthymia disorders is heavier, the ill degree of dysthymia disorders core symptom is heavier, it is better to take behind the thymoleptic drug effect of thymoleptic, and thymoleptic are better to the drug effect of dysthymia disorders core symptom; When 5-HTT13C/T (rs2054847) pleomorphism site genotype is the homozygous or 13CT heterozygous of 13CC, predict that the ill degree of individual dysthymia disorders is lighter, the ill degree of dysthymia disorders core symptom is lighter, it is relatively poor to take behind the thymoleptic drug effect of thymoleptic, and thymoleptic are relatively poor to the drug effect of dysthymia disorders core symptom.
In method of the present invention, can use the following difference foranalysis of nucleic acids technology that is selected from that comprises: polymerase chain reaction (PCR), polymerase chain reaction-restriction fragment length polymorphism, PCR-alleles-specific oligonucleotide probe method, PCR-sequence specific oligonucleoside acid system, sequencing, PCR-sequence specific primers method, the PCR-fluorescent method, the PCR finger-printing method, oligonucleotide connects to be analyzed, the detection method of fluorescent energy resonance transfer, biochip, nucleic acid chip, mass-spectrometric technique, genescan, single strand conformation polymorphism, denaturing gradient gel electrophoresis, enzyme or chemical mispairing patterning method, the Taqman biological detecting method, nanotechnology.
PCR, PCR-RFLP, biochip, sequencing, genescan genotype detection method are the conventional methods of using of those skilled in the art.The Taqman technology is a kind of method of using fluorescence technique to carry out real-time quantitative PCR.Biochip is meant methods such as adopting the synthetic or micro-sampling of Hiroshima original position, with the large number of biological macromole such as nucleic acid fragment, peptide molecule even tissue slice, biological samples such as cell solidify in upholder in an orderly manner (as slide, silicon chip, polyacrylamide gel, carriers such as nylon membrane) surface, form intensive two-dimentional molecular arrangement, then with the molecular hybridization that hits of the biological sample to be measured of mark, by specific instrument such as laser confocal scanning instrument or electric charge coupling photographic camera (CCD) intensity of hybridization signal is carried out fast, parallel, check and analysis efficiently, thereby the quantity of target molecule or quality in the judgement sample.Nanotechnology is utilized the gold nano grain of functionalization, and the particle diameter scope is at 10-40nm.Specific oligonucleotide sequence is attached to the gold nano grain surface, makes signal obtain richness and amplification by specific molecular hybridization, specific fluorescent signal system or other Color Appearance Systems, thereby obtains genotypic information.Wherein preferred detection method is PCR, PCR-RFLP, Taqman technology, biochip, nucleic acid chip or nanotechnology.The present invention is not to be qualification for detection method for the explanation of the genotypic detection method of pleomorphism site, any those skilled in the art adopt conventional biotechnological means to predict the ill degree of individual dysthymia disorders by detecting pleomorphism site genotype of the present invention, the method of thymoleptic drug effect all belongs to content of the present invention, can further include to adopt conventional biotechnological means to predict the ill degree of individual dysthymia disorders in the biological sample by the pleomorphism site that the indirect reflection of the difference that detects genotypic transcription product of pleomorphism site functional type of the present invention and/or expression product is associated, the method of thymoleptic drug effect.
In the present invention, described biological sample is selected from: blood sample, humoral sample, tissue sample and culturing cell, preferred, described sample is a blood sample.Wherein blood sample comprises peripheral blood cells, white corpuscle, serum etc., humoral sample comprises urine, saliva, tissue juice, cerebrospinal fluid, body cavity transudate etc., and tissue sample comprises oral mucosa examination son, hair, skin, biopsy, organizes sample secretory product, fecal matter sample etc.
The present invention also provides and has been used to predict the ill severity of individual dysthymia disorders, the ill severity of dysthymia disorders core symptom, the drug effect of predicting thymoleptic and the thymoleptic test kit to the drug effect of dysthymia disorders core symptom.In test kit provided by the invention, when 5-HTT13C/T (rs2054847) pleomorphism site genotype is that 13TT is when homozygous, predict that the ill degree of individual dysthymia disorders is heavier, the ill degree of dysthymia disorders core symptom is heavier, it is better to take behind the thymoleptic drug effect of thymoleptic, and thymoleptic are better to the drug effect of dysthymia disorders core symptom; When 5-HTT13C/T (rs2054847) pleomorphism site genotype is the homozygous or 13CT heterozygous of 13CC, predict that the ill degree of individual dysthymia disorders is lighter, the ill degree of dysthymia disorders core symptom is lighter, it is relatively poor to take behind the thymoleptic drug effect of thymoleptic, and thymoleptic are relatively poor to the drug effect of dysthymia disorders core symptom.
In test kit provided by the invention, can comprise the genotypic polymorphism parting oligonucleotide of 13C/T pleomorphism site probe that is used for detection of biological sample 5-HTT gene and the reaction system that detects.Wherein, oligonucleotide probe can be specifically and the nucleic acid hybridization of the 13C/T pleomorphism site that contains described 5-HTT gene.Oligonucleotide probe preferably comprises the probe shown in SEQ ID No.3 and the SEQ ID No.4.
The sequence of allele-specific probe is:
VIC-5’TCAGGCAC
GACATTT3’-NFQ(SEQ?ID?No.3)
FAM-5’TCAGGCAC
AACATTT3’-NFQ(SEQ?ID?No.4)
In test kit provided by the invention, can comprise be used for amplification contain 5-HTT gene the 13C/T pleomorphism site 5-HTT gene fragment Auele Specific Primer, can specific recognition and the restriction enzyme and the damping fluid thereof that cut off described site specific nucleic acid sequence.Wherein, restriction enzyme is preferably AvaII.In the mentioned reagent box, can further include nucleic acid extracting reagent, PCR reaction reagent and positive control template.Wherein, the PCR reaction reagent contains four kinds of deoxymononucleotides, hot resistant DNA polymerase and reaction buffers thereof; The positive control template comprises the PCR product, cloning nucleic acid, cloned plasmids of homozygous, the heterozygous of the 13C/T pleomorphism site that contains the 5-HTT gene and/or homozygous sequence, directly synthetic nucleotide sequence or genomic dna.
Primer sequence is:
Forward primer: 5 ' GTAATAAAATATTATTTATTTTGATTTTGGCTCCTGCTG3 ' (SEQ IDNo.1)
Reverse primer: 5 ' AGAAACTCAATGACTGTGCATCAAGA3 ' (SEQ ID No.2)
The present invention provides a kind of gene chip again, wherein comprises the genotypic polymorphism parting oligonucleotide of the pleomorphism site probe that is used to measure 5-HTT; Wherein said gene chip is preferably the DNA chip.
Should be understood that, all technical schemes of the present invention are not subject to any theory, the present invention is not to be qualification for detection method for the explanation of the detection method of loci gene type yet, any those skilled in the art adopt conventional biotechnological means to predict with the ill degree of dysthymia disorders by detecting pleomorphism site genotype of the present invention, the antidepressant drug drug effect is that other inventions of core all belong to content of the present invention, can further include to adopt conventional biotechnological means to predict the ill degree of dysthymia disorders by the pleomorphism site that the indirect reflection of the difference that detects genotypic transcription product of pleomorphism site of the present invention and/or expression product is associated, the example of antidepressant drug drug effect.
Following embodiment has carried out further instruction to the present invention, but it will be appreciated by those skilled in the art that all embodiment just in order to understand the present invention better, and it all is not in order to limit content of the present invention going up in all senses.
Embodiment
Embodiment 1 measures the effect of 5-HTT gene 13C/T polymorphism site genotype estimation depression severity
One. research object
1 patient source:
The whole nation 12 tame spiritual section hospitals and general hospital polyclinic's psychiatric department or Psychology Dept. out-patient.
2 include standard in:
The male and female patient of age 〉=18 year old; According to Americanism obstacle diagnosis and the 4th edition (Diagnosis and statistic manual for mental disorder-IV of statistic handbook, DSM-IV) be diagnosed as major depression (major depressive disorder), previously do not have manic or the hypomania history.The row that simple dysthymic disorder (dysthymia) is not being included in; HAMD17 item total points 〉=16 minutes; According to investigator's viewpoint, patient must can cooperate all research steps.
3 exclusion standards:
● psychiatric history previously: 1) can not except other organic or drug-induced secondary depression or two-way affective disorder; 2) serious introgression, stuporous state, obsession, disease of eating too much at one meal or other serious mental disorder medical histories are arranged; 3) wine or other substance depilatories or abuse evidence person are arranged.
● other medical histories: 1) back 1 year convalescence of operation; 2) there were gastrointestinal tract disease such as stomach ulcer, chronic diarrhoea, liver and gall diseases (chronic hepatitis, liver cirrhosis, Biliary Calculi etc.), primary chronic nephropathy, renal insufficiency etc. to influence the patient of normally absorption of fluoxetine, metabolism, distribution and excretory function in 1 year; 3) the serious brain traumatism history is arranged; Or other serious complication are arranged, as organic disease of chronic progressive externals such as cerebral apoplexy, heart trouble, diabetes, cardiac insufficiency or deterioration etc.; 4) clear and definite body or neural system serious disease person are found in laboratory or physical examination.
● history of medications: 1) accepted ECT (ECT) in nearest six months, or long lasting antipsychotics of life-time service or fluoxetine; 2) once accepted system's anti depressant medication in (therapeutic dose is more than 4 weeks) in nearest three months; 3) recently around in took any antipsychotics or antidepressant drug, no matter dosage is much or the course of treatment how long; 4) used oxidase inhibitor in nearest two weeks; 5) must merge the medicine person who uses medicine maybe may sway the emotion for a long time with central nervous system effect;
6) to fluoxetine allergy
Childbearing history: gestation or lactating women.Do not take at present effective contraceptives of medically approving thereby might conceived person
Two research methods
1 evaluation tool and method:
(1) Case definition: the 4th edition (American PsychiatricAssociation.Diagnostic and Statistical Manual Disorder (DSM-IV) .4th of Americanism obstacle diagnosis and statistic handbook, Washington:psychiatric Association, 1994.320-327) Case definition of major depression.
(2) diagnosis scale: genetics research deagnostic test (Nurnberger J.J., Blehar, M., Kaufmann, C., et al.Diadgnostic interview for genetic studies; Rationale, uniquefeatures and training; NIMH Genetics Initiative, the Arch.Gen.Psychiatry.1994) third edition
(3) severity measuring scale (Zhang Mingyuan chief editor psychiatric department scale evaluation handbook, Hunan science tech publishing house, 1998): 17 versions of Hamilton depression scale (HAMD)
The HAMD scale is with syndrome fall into 5 types the accordingly factor (Cleary M, Guy W.Factoranalysis of the Hamilton depressionscale.Drugs Exp Clin Res1975; 1:115-20.), comprise retardation factor (HAMD clauses and subclauses 1,7,8,14); The sleep symptom factor (HAMD clauses and subclauses 4,5,6); The Maier symptom factor (HAMD clauses and subclauses 1,2,7,8,9,10); The anxiety factor (HAMD clauses and subclauses 10,11,12,13,15,17); The core factor (HAMD clauses and subclauses 1,2,3,7,8); The verification factor=HAMD total points-anxiety factor branch-sleep factor branch (HAMD clauses and subclauses 1,2,3,7,8,9,14,16).。
Clinical general curative effect measuring scale CGI (Clinical Global Impression): the versions in 1976 that are America NI MH revision.Disease severity SI (severity of illness) adopts 8 grades of scoring systems of 0-7 minutes, according to concrete patient's the state of an illness and other similar patients' comparisons of same research, makes evaluation.
(4) epidemiology questionnaire:
All participators answered the epidemiology questionnaire at the 1st day, content comprises: history of disease, have or not negative life events in 3 months or in 3 months to 1 year; Occupational history: comprise employment experience, job specification, daily vocational activity and occupational exposure etc.; Living habit: physical activity, rhythm of life, sleep quality, smoking and passive smoking situation, drink and drink tea situation etc.; Daily meals situation: food habits, every day vegetables, fruit, meat, nonstaple food absorption situation etc.
2 research process:
(1) finishes Hamilton depression scale and clinical global impression (CGI) evaluation on the 1st day through the qualified patient of screening, finish the baseline evaluation of the side effect scale that occurs in the treatment, genetics research deagnostic test (DIGS) and epidemiology questionnaire etc.
(2) standard compliant patient enters the pharmacological agent phase.To oral with the fluoxetine dispersible tablet, 20 milligrams of every days, the morning or the morning are oral.Treatment 6 weeks of observation period.Treatment the day before yesterday, treat first and second, respectively carry out Clinical Follow-up four, six weekends one time.
(3) blood-sample withdrawal and genotype detection: gather patient's vein EDTA anticoagulated whole blood 10ML, the blood sampling back is mixing fully, packing, and it is frozen to be put in-20 ° of C, finishes DNA extraction and gene type assay in 3 months.
Three laboratory parts
1 extracts the genomic dna of host cell:
(1) add the 30ml erythrocyte cracked liquid in the blood, slowly shake up, room temperature left standstill 10 minutes, during, shake for several times, thoroughly splitting erythrocyte;
(2) in 4 ℃, 2000 leave the heart/minute, 10 minutes, remove supernatant, the white corpuscle that will precipitate is broken up on the oscillator in rotation, adds proteolytic enzyme 40ul, RNA enzyme 50ul, shakes up, and adds write cell lysis buffer and puts 15ml, 37 ℃ of water-baths of mixing were taken out after 20 minutes, put in the cold water;
(3) add cold albumen precipitation liquid 4ml, be placed on-20 ℃ of refrigerators 5 minutes behind the mixing, take out in 4 ℃, 3000 rev/mins centrifugal 10 minutes.Supernatant liquor poured into slowly shake in the 50ml centrifuge tube that oneself has added the 15ml Virahol for several times, separate out to the DNA floss;
(4) the DNA floss of separating out is moved to another 1.5ml and has packed on the 75% alcoholic acid filter paper, make evaporate dried;
(5) add DNA hydrating fluid 1.5ml, put shaking table, shaken over night, standby;
(6) mensuration of DNA concentration adopts ultraviolet spectrophotometry, measures the OD value under two wavelength of 260nm and 280nm respectively, is DNA concentration with OD260nm * 50 income values.And with OD260nm/OD280nm ratio estimation DNA purity.
2 use the Taqman method to detect the 13C/T pleomorphism site genotype of 5-HTT gene
(1) with PCR instrument amplification 5-HTT functional gene polymorphic site and flanking sequence thereof, in 5ul PCR reaction system, contain genomic dna 10ng, 2.5ul Taqman2X Universal PCR Master MixNo AmpErase UNG (composition comprises: AmpliTaq Gold DNA Polymerase, dNTPs withDutp, Passive Reference, with the damping fluid of optimizing), and the forward primer of 0.72uM, each 0.16uM. of allele-specific probe of the reverse primer of 0.72uM and two sections band fluorescence report groups
Primer sequence is
Forward primer: 5 ' GTAATAAAATATTATTTATTTTGATTTTGGCTCCTGCTG3 ' (SEQ ID No.1)
Reverse primer: 5 ' AGAAACTCAATGACTGTGCATCAAGA3 ' (SEQ ID No.2)
The sequence of allele-specific probe is:
VIC-5’TCAGGCAC
GACATTT3’-NFQ(SEQ?ID?No.3)
FAM-5’TCAGGCAC
AACATTT3’-NFQ(SEQ?ID?No.4)
The PCR reaction conditions: circulate in advance 95 ° C10 minute; 92 ° of C15s, 60 ° C1 minute the circulation 50 times.
(2) on 7900 type quantitative real time PCR Instruments, detect fluorescence information, carry out the genotype result and judge:
The PCR plate of finishing the PCR reaction is put on the 7900 type quantitative real time PCR Instruments, is selected for use " AllelicDiscrimination " program, scan judgement with the result:
The genotype of sending FAM fluorescence person is the T/T homozygote;
The genotype of sending VIC fluorescence person is the C/C homozygote;
The genotype of sending two kinds of fluorescence persons is the C/T heterozygote.
Four statistical procedures
Use the SPSS11.5 statistical package to analyze.Adopt the chi-square analysis genotype frequency whether to meet the Hardy-Weinberg equilibrium law.Descriptive statistic: continuous variable is done the analysis that whether meets normal distribution, do not meet and at first adopt data-switching, make it to meet normal distribution.Inferential statistics: P value: during statistical calculations, provide concrete P value.P＜0.05 expression has statistical significance.Measurement data: t check, one-way analysis of variance and multiple regression analysis.Enumeration data: adopt chucking method, proofread and correct chi square test.
The result
1 5-HTT allelotrope and genotype distribution situation
The selected research of baseline period 761 routine patients, wherein the blood sample of 425 examples (56%) has carried out the DNA detection genotype.5-HTT allelotrope and genotype distribution situation are referring to table 1.Wherein CC genotype patient's ratio is low, CC and CT genotype patient is merged into contain the allelic one group of patient of C, is called for short C+, and the genotypic patient of TT does not contain C allelotrope, abbreviates C-as.Carry out Hardy-Weinberg balance check: χ
2=0.839, P=0.36 meets the Hardy-Weinberg balance, and the colony of sample from genetic equilibrium is described.
Table 1 5-HTT genotype and gene frequency (N=425)
The selection of genetic model in the 2 5-HTT gene type assays
Three genotypic patients are respectively TT type (n=278) in the average of baseline period HAMD total points: 23.19 ± 5.23, and CT type (n=135): 21.98 ± 4.62 and CC type (n=12): 20.83 ± 4.06.TT type and CT type patient HAMD total points are relatively, difference has significance (to proofread and correct sex, age, this course of disease, single/recurrence and having or not in 1 year after the factors such as life event, P=0.043), CT type and CC type patient HAMD total points are relatively, therefore difference does not have significance, and (correction factor is the same, P=0.425), adopts the dominant inheritance model with the CT type with CC type patient merges into one group (C+ type) and TT type (C one type) patient compares.
3 baseline period data and 5-HTT genotype compare:
Enumeration data adopts chi square test, and measurement data adopts independent sample t check (using mean ± standard deviation to represent), and wherein this course of disease, attack times and CGI-SI adopts nonparameter test, uses median (minimum value～maximum value) expression.Baseline period records genotypic patient's 425 examples of 5-HTT.Patient's sex, age of onset this time, age of onset first, attack times, nationality, marital status, the length of education enjoyed, single or recurrence, this course of disease, premorbid has or not indifference between the 5-HTT genotype such as negative life events in 3 months; And frequency, CGI-SI mark, HAMD total points that the TT genotype occurs negative life events in 1 year at premorbid be higher than and contain C+ type patient, and statistical significance is arranged.Wherein TT genotype patient the negative life events ratio was arranged in 1 year is 1.86 times (95%CI:1.23-2.83) that the C+ genotype has the negative life events ratio.See Table 2.
Table 2 baseline period data and genotype
"-" expression nonparameter test
4 baseline period HAMD total points and genotypic relation
Compare the difference between the depressed severity of different genotype patients with depression.Owing to use regression analysis consistent with t check analysis methods and results, use simultaneously regression analysis be convenient to we with correcting variable after data compare, so what show in form is that 2 groups of data of proofreading and correct front and back are adopted in regression analysis.Correction factor is the same.The difference of different genotype patient's HAMD total points has statistical significance.TT genotype patient's HAMD total points is higher than the genotypic patient of C+ significantly.See Table 3.
This presentation of results, this gene locus polymorphism is relevant with the severity of dysthymia disorders, and TT genotype patient dysthymia disorders is even more serious.
The 5 baseline period factors are divided and genotypic relation
Compare the difference between each syndrome severity of different genotype patients with depression.Analytical procedure is the same, and different genotype patient's retardation factor, the core factor, Maier factor mark have statistical significance.We set up the verification factor according to the result, verification factor mark=HAMD total points-(anxiety factor mark+sleep factor mark), and the difference of icp gene type, and analytical procedure is the same.TT genotype patient is higher than the genotypic patient of C+ significantly at retardation factor, the core factor, the Maier factor and verification factor mark.See Table 3.
Each clauses and subclauses mark of 6 baseline period HAMD and genotypic relation:
For the The data independent sample t check that meets normal distribution, for analyzing through the The data nonparameter test that does not still meet normal distribution after the conversion.The result shows TT genotype patient's HAMD scale clauses and subclauses 1 depressive mood, and clauses and subclauses 2 senses of guilt and clauses and subclauses 7 job interests are lost the patient that these 3 clauses and subclauses scorings are higher than the C+ type.See Table 4.
Our genotypic patient of TT genotype and C+ that studies show that compares, and at age of onset, aspects such as sex do not have statistical difference; Yet negative life events is many in 1 year of TT genotype patient report, and HAMD total points and CGI-SI mark height show that the depressive symptom evaluation is serious; TT genotype patient retardation factor, the mark height of the core factor and the Maier factor shows to depressive mood, sense of guilt and job interest forfeiture in the evaluation of the severity of single symptom obviously.
Some clauses and subclauses has overlapping in 5 factors of Shi Yonging under study for action, for example the retardation factor and the core factor are owned clauses and subclauses 1,7,8 together, comprised the evaluation of anxiety aspect clauses and subclauses in the Maier factor, therefore the result to factorial analysis need carry out Comprehensive Assessment in conjunction with the analysis of the verification factor.What studies show that originally that the TT genotype mainly influences is the core symptom of depressive patient, and relative with the influence of anxiety aspect less to sleep.
5-HTT 13 C/T (rs2054847) gene pleiomorphism can be modified the perception of depressive patients to negative life events, modify the severity of dysthymia disorders, and mainly be the core symptom of modifying dysthymia disorders, the severity of symptoms such as promptly subjective depression, hebetude and little with symptom relationships such as anxiety, sleeps.TT homozygote depressive patients is tended to suffer from even more serious, and more typical dysthymia disorders.
Table 3 baseline period HAMD total points, the factor are divided and the genotype multiple regression analysis
* correction factor: sex, at the age, this course of disease had or not life event in 1 year, single/recurrence etc.
Table 4 baseline period HAMD clauses and subclauses mark and genotype concern t check and * nonparameter test
The relation of embodiment 2 fluoxetine curative effect of medication and five hydroxytryptamine transporter gene pleiomorphism
One research object
1 patient source: see embodiment 1.
2 research medicines: fluoxetine (Fluoxetine), Lilly Co., Eli. produces, the trade(brand)name prozac.Lot number: A022974, A042635, A057046, A060206, A077246), (every day 1 time, each 1, every 20mg, totally 43 days), fixed dosage.
3 clinical study processes:
(1) research process: finished Hamilton depression scale and clinical global impression (CGI) evaluation on the 1st day through the qualified patient of screening, finish the baseline evaluation of side effect (TESS) scale that occurs in the treatment, genetics research deagnostic test (DIGS) and epidemiology questionnaire etc.; Oral fluoxetine a slice of physician guidance patient (20mg) is introduced the characteristics of pharmacological agent and common side reaction to patient.Reservation is followed up a case by regular visits to, and instructs to fill in and follows up a case by regular visits to diary, provides all medicines and follows up a case by regular visits to diary.
The 2nd～42 day patient every day is one of the oral fluoxetine of daystart, and fills in the diary of taking medicine.The patient checks (2 days the allowed band in front and back can be arranged) in the back the 8th, 15,29,43 day of beginning to take medicine therebetween to hospital, is carried out inspections such as HAMD, CGI, TESS by the doctor.The doctor checks patient's the situation of taking medicine, the concurrent medicine of taking in week that puts down.Simultaneously, during the pharmacological agent weekly phone follow up a case by regular visits to 2 times, supervise patient to take medicine on time, fill in and follow up a case by regular visits to record, the disposal that monitoring side reaction and symptom lapse to.Further consultation the day before yesterday, carry the packing box of the medicine of taking medicine diary and the medicine that does not take and having taken when reminding patient's further consultation next day and further consultation by expeditor's phone, be used to check patient's the situation of taking medicine.
(2) merge the use medicine: during fluoxetine pharmacological agent, must not merge and use other antidepressant drug, the various medicines that mainly act on central nervous system are used in nonjoinder as far as possible, comprise antipsychotics, antidepressant drug, antimanic agents, antiepileptic drug etc.For the patient who occurs obvious somnopathy in the treatment, can short-term (being no more than for 2 weeks) merge use the roller west dissolve (＜6mg), clonazepam (＜6mg), the nitro west dissolve (＜15mg) or estazolam (＜3mg) wherein a kind of.This type of medicine is used in nonjoinder as far as possible in last two weeks of treatment, in order to avoid influence the evaluation of net result.
(3) treatment of special situation between follow-up period: the doctor should notice that the state of an illness of observing the patient lapses in the research process, in case find severe complications, sb.'s illness took a turn for the worse or whenever serious side reaction or patient proposes to withdraw from observation, all should in time stop research.Especially at first two weeks of treatment, the doctor should note observing the patient and have or not introgression and other serious mood disorderss in the research.Should explain purpose, the especially patient's of patient's state of an illness and this research suicide idea and behavior to family numbers of patients in detail.Take the reactions such as gastrointestinal discomfort, anxiety, excitation, insomnia, sexual dysfunction or fash that medicine itself may cause.Look severity by the doctor and in time handle, and on case report form (CRF), carry out record.If the patient exists and to miss medicine, or allow to take situation such as other drug without the doctor, need conscientiously write down consumption, Time of Administration and medicine name etc., miss continuously and (do not contain 3 days) more than 3 days or accumulation misses and (do not contain 8 days) more than 8 days, withdraw from research.Dosage the scheme prescribed dose 80～120% between patient be the good person of compliance.The doctor should carry out strict tracing study to patient's the situation of taking medicine.Be included in when following up a case by regular visits at every turn and introduce administrated method and the common side reaction of medicine in detail, allow will take medicine back remaining empty medicine box and bubble plate of patient reclaim and examination, supervise patient to remember and follow up a case by regular visits to diary etc. to patient.
Two data typing and statistical procedures
Use the SPSS11.5 statistical package to analyze.Descriptive statistic: continuous variable is done the analysis that whether meets normal distribution, do not meet and at first adopt data-switching, make it to meet normal distribution.Inferential statistics: P value: during statistical calculations, provide concrete P value.P≤0.05 expression has statistical significance.Measurement data: one-way analysis of variance and multiple regression analysis.Enumeration data: adopt chucking method, proofread and correct chi square test, Logistic regression analysis etc.
602 examples (79.1%) have been finished treatment in 43 days and have been followed up a case by regular visits to assessment among the baseline period 761 routine patients.
Three genotype and therapeutic effect relationship
1 curative effect evaluation criteria:
Clauses and subclauses mark when (1) following up a case by regular visits to, factor mark and HAMD total points.
(2) subtract branch and subtract the branch rate: the baseline mark deducts when following up a case by regular visits to the fractional score as subtracting branch.Subtracting the branch rate is (subtracting branch/baseline mark) * 100%.
(3) alleviate (remission): HAMD marked＜=7 fens as clinical remission standard (Keller MB.Remission versus response:The new gold standard of antidepressant care.J Clin Psychiatry2004 when 6 weeks of anti-depressant therapy finished; 65:53-59).
(4) effectively (respond): subtract the branch rate before and after the HAMD treatment when 6 weeks of anti-depressant therapy finish and descend 〉=50% as effective (Stassen HH, Angst J, Delili-Stula A.Severity at baseline and onset ofimprovement in depression:meta-analysis of imipramine and moclobemide versusplacebo Eur Psychiatry1994; 9:129-136).
Total points subtracted branch and genotypic relation when 2 researchs finished:
Proofread and correct factors such as sex, age, this course of disease, single-shot/recurrence, baseline HAMD mark, life event, carry out regression analysis.(43d) TT genotype patient HAMD subtracted branch (10.18 ± 5.74 when regression analysis was presented at treatment 29d and end before proofreading and correct, subtract branch (8.81 ± 5.65 13.91 ± 6.20) be higher than C+ genotype HAMD, 12.41 ± 5.68), this trend still exists after proofreading and correct above-mentioned variable.See Table 5.
3 inquire into the efficient and genotypic relation when finishing of studying:
Proofread and correct sex, age, the inferior course of disease, single-shot/recurrence, life event etc., carry out the Logistic regression analysis.When research finishes (43d), TT genotype patient is efficient to be 74.7% (162/217), and the C+ genotype is efficient to be 69.6% (78/112), and efficient TT type presents the trend that is higher than C+ type patient.
4 inquire into remission rate and the genotypic relation when finishing studied:
Carry out the Logistic regression analysis, correcting variable is sex, age, this course of disease, single-shot/recurrence, life event in 1 year, baseline HAMD mark etc.When research finishes (the 43rd day), TT genotype patient remission rate (48.3%, 44/112) be 1.68 times of remission rate (39.3%, 105/217) of C+ type patient (R2=0.096, P=0.036).
5 analysis factors subtract branch and genotypic relation:
When research finishes (43d), TT genotype patient's core, sluggishness and the Maier factor subtract branch and are higher than C+ type patient, and statistical significance is arranged; Wherein to subtract the difference of branch be that 15d from treatment begins to occur for the core factor and the Maier factor, illustrate the core symptom group who relates to dysthymia disorders factor subtract the morning that branch occurs on the TT genotype.See Table 6,7.
The 6 verification factors and genotypic relation:
The verification factor that we set up in first part research (the HAMD total points deducts the score value of the sleep and the anxiety factor) subtracts score value when calculating it and following up a case by regular visits to.The verification factor subtracts branch when research finishes: the TT type is 7.75, the C+ type is 6.47, the research genotype subtracts the influence of branch to the factor, correcting variable is the same, show that the TT type verification factor subtracts branch and is higher than C+ type patient (t=-2.413 significantly, P=0.016), the core factor that has proved the depression of TT type patient except that sleep and anxiety subtracts branch significantly.
After patients with depression uses Fluoxetine in Treatment, TT genotype patient's total points subtracts branch more than C+ genotype patient, and efficient and remission rate is significantly higher than C+ genotype patient, illustrate TT genotype patient take fluoxetine after doing well,improving be better than the C+ genotype patient who takes same medicine.In the time of 43 days, the core factor of taking the TT genotype patient of fluoxetine subtracts branch, retardation factor and subtracts branch and the Maier factor and subtract branch and be higher than C+ type patient, statistical significance is arranged, illustrate that TT genotype patient takes behind the fluoxetine improvement of symptom and shows that mainly core symptom improves, distinguish not obvious for the improvement of corporality symptoms such as sleep, anxiety and the result of treatment of taking the C+ genotype patient behind the fluoxetine.TT genotype patient compares with C+ genotype patient, to subtract the difference of improving that branch and the Maier factor subtract branch be that from treatment the 15th day begins to occur to the core factor between follow-up period, illustrate the core symptom group who relates to dysthymia disorders factor subtract the morning that branch occurs on TT genotype patient, show that TT genotype patient uses SSRI class antidepressant drug just can obviously improve the dysthymia disorders core symptom in early days in treatment.Illustrate that the curative effect of SSRI class thymoleptic Fluoxetine in Treatment of Depression is subjected to the modification of 5-HTT13C/T (rs2054847) polymorphism.General curative effect significantly improves after the TT genotype patient, and mainly is reflected in significantly quickening of dysthymia disorders core symptom improvement, final raising evident in efficacy.
The HAMD total points subtracts branch and genotype between table 7 follow-up period
Correcting variable: sex, age, this course of disease, single/recurrence, life event in 1 year, baseline period HAMD total points
The factor subtracts branch and genotype between table 8 follow-up period
Positive variable: sex, the age, this course of disease, single-shot/recurrence, life event in 1 year, baseline mark TT genotype is compared * P＜0.05 with the C+ genotype
The factor subtracted the multiple regression analysis of branch when table 9 TT genotype and C+ genotype were followed up a case by regular visits to 43d
Correction factor: sex, age, this course of disease, baseline mark, life event etc. in 1 year
Embodiment 3: test kit and implementation method thereof
Present embodiment provides a kind of test kit of predicting the dysthymia disorders occurrence risk illustratively, and is composed of the following components:
(1) erythrocyte cracked liquid: contain NH4Cl, KHCO3, EDTA;
(2) write cell lysis buffer: contain Proteinase K (Sigma), RNase A (Promega), NaCl, 5 * Tris, EDTA, SDS;
(3) albumen precipitation liquid: 7.5M amine acetate (pH7.4)
(4) nucleic acid storage liquid: 1M Tutofusin tris-hydrochloric acid (Tris-HCl, pH8.0);
(5) PCR reaction mixture: 1.0ml[100mM Tris-HCl, 100mM KCl, pH8.3 (20 ° of C)]; 5.0 μ M MgCl2 (Applied Biosystem); Each 0.4mM dATP, dCTP, dGTP, dTTP, the aseptic two preparation of 10 water, pH7.0 (BioBasic Inc.) of steaming; The pleomorphism site genotype detection primer of PRCP gene (worker's biotechnology company limited is given birth in Shanghai);
(6) Taq archaeal dna polymerase (Applied Biosystems) (5U/ μ l): preserve damping fluid: 20mMTris-HCl (pH8.0), 100mM KCl, 0.1mM EDTA, 1mM DTT, 0.5%NP40,0.5% (v/v) Tween20,50% glycerine;
(7) positive plasmid: contain the individual whole blood sample of pleomorphism site heterozygous genes type of 5-HTT gene, perhaps contain the plasmid of the pleomorphism site heterozygous genes type of 5-HTT gene;
(8) negative control: through the distilled water of DNAase I (Promega) processing;
(9) restriction endonuclease buffer system (10 *);
(10) restriction enzyme;
(11) PCR water;
(12) 10 * electrophoresis sample-loading buffers: 0.25% bromjophenol blue (Sigma), 40% (w/v) aqueous sucrose solution.The mentioned reagent box can for example be used to measure the 13C/T pleomorphism site genotype of 5-HTT gene:
1 extracts the genomic dna of host cell:
(1) add the 30ml erythrocyte cracked liquid in the blood, slowly shake up, room temperature left standstill 10 minutes, during, shake for several times, thoroughly splitting erythrocyte;
(2) in 4 ℃, 2000 leave the heart/minute, 10 minutes, remove supernatant, the white corpuscle that will precipitate is broken up on the oscillator in rotation, adds proteolytic enzyme 40u1, RNA enzyme 50ul, shakes up, and adds write cell lysis buffer and puts 15ml, 37 ℃ of water-baths of mixing were taken out after 20 minutes, put in the cold water;
(3) add cold albumen precipitation liquid 4ml, be placed on-20 ℃ of refrigerators 5 minutes behind the mixing, take out in 4 ℃, 3000 rev/mins centrifugal 10 minutes.Supernatant liquor poured into slowly shake in the 50ml centrifuge tube that oneself has added the 15ml Virahol for several times, separate out to the DNA floss;
(4) the DNA floss of separating out is moved to another 1.5ml and has packed on the 75% alcoholic acid filter paper, make evaporate dried;
(5) add DNA hydrating fluid 1.5ml, put shaking table, shaken over night, standby;
(6) mensuration of DNA concentration adopts ultraviolet spectrophotometry, measures the OD value under two wavelength of 260nm and 280nm respectively, is DNA concentration with OD260nm * 50 income values.And with OD260nm/OD280nm ratio estimation DNA purity.
2 use the Taqman method to detect the C/T pleomorphism site genotype of 5-HTT (five hydroxytryptamine transporter) gene
(1) with PCR instrument amplification 5-HTT functional gene polymorphic site and flanking sequence thereof, in 5ul PCR reaction system, contain genomic dna 10ng, 2.5ul Taqman2X Universal PCR Master Mix No AmpErase UNG (composition comprises: AmpliTaq Gold DNA Polymerase, dNTPs with Dutp, Passive Reference, with the damping fluid of optimizing), and the forward primer of 0.72uM, each 0.16uM. of allele-specific probe of the reverse primer of 0.72uM and two sections band fluorescence report groups
Primer sequence is
Forward primer: 5 ' GTAATAAAATATTATTTATTTTGATTTTGGCTCCTGCTG3 ' (SEQ ID No.1)
Reverse primer: 5 ' AGAAACTCAATGACTGTGCATCAAGA3 ' (SEQ ID No.2)
The sequence of allele-specific probe is:
VIC-5’TCAGGCAC
GACATTT3’-NFQ(SEQ?ID?No.3)
FAM-5’TCAGGCAC
AACATTT3’-NFQ(SEQ?ID?No.4)
The PCR reaction conditions: circulate in advance 95 ° C10 minute; 92 ° of C15s, 60 ° C1 minute the circulation 50 times.
(2) on 7900 type quantitative real time PCR Instruments, detect fluorescence information
The PCR plate of finishing the PCR reaction is put on the 7900 type quantitative real time PCR Instruments, is selected for use " AllelicDiscrimination " program, scan judgement with the result:
The genotype of sending FAM fluorescence person is the T/T homozygote;
The genotype of sending VIC fluorescence person is the C/C homozygote;
The genotype of sending two kinds of fluorescence persons is the C/T heterozygote.
According to genotype detection prediction of result dysthymia disorders occurrence risk as described in the embodiment 1.
Sequence table
＜110〉Hua'anfo Medicine Research Center Co., Ltd., Beijing
＜120〉purposes, method and the test kit of polymorphism site genotype estimation depression generation and prognosis
<130>122212223
<160>4
<170>PatentIn?version3.3
<210>1
<211>39
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＜213〉synthetic
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<213>GTAATAAAATATTATTTATTTTGATTTTGGCTCCTGCTG
<400>2
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＜213〉synthetic
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Claims (1)
1. be used to predict the test kit of ill degree of dysthymia disorders or fluoxetine drug effect, this test kit comprises the genotypic oligonucleotide probe of 13C/T pleomorphism site that is used for detection of biological sample 5-HTT gene, its probe sequence is shown in SEQ ID No.3 and SEQ ID No.4, and the reaction system that detects, this reaction system also contains the Auele Specific Primer of the 5-HTT gene fragment that being useful on increases contains the 5-HTT pleomorphism site, and this primer sequence is shown in SEQ ID NO.1 and SEQ ID NO.2; The 13C/T pleomorphism site of wherein said 5-HTT gene is rs2054847, when the 5-HTT 13 C/T pleomorphism site genotype that detect are 13TT when homozygous, predicts that the ill degree of individual dysthymia disorders is heavier, to take behind the fluoxetine drug effect of fluoxetine better; When 5-HTT 13 C/T pleomorphism site genotype are the homozygous or 13CT heterozygous of 13CC, predict that the ill degree of individual dysthymia disorders is light, to take behind the fluoxetine drug effect of fluoxetine relatively poor.
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